Critical Reviews in Food Science and Nutrition, 50:515532 (2010) Copyright C Taylor and Francis Group, LLC ISSN:

1040-8398 DOI: 10.1080/10408390802565889

Factors Influencing the Chemical Stability of Carotenoids in Foods

CAITLIN S. BOON, D. JULIAN MCCLEMENTS, JOCHEN WEISS, and ERIC A. DECKER
Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, Massachusetts 01003

In recent years, a number of studies have produced evidence to suggest that consuming carotenoids may provide a variety of health benets including a reduced incidence of a number of cancers, reduced risk of cardiovascular disease, and improved eye health. Evolving evidence on the health benets of several carotenoids has sparked interest in incorporating more carotenoids into functional food products. Unfortunately, the same structural attributes of carotenoids that are thought to impart health benets also make these compounds highly susceptible to oxidation. Given the susceptibility of carotenoids to degradation, particularly once they have been extracted from biological tissues, it is important to understand the major mechanisms of oxidation in order to design delivery systems that protect these compounds when they are used as functional food ingredients. This article reviews current understanding of the oxidation mechanisms by which carotenoids are degraded, including pathways induced by heat, light, oxygen, acid, transition metal, or interactions with radical species. In addition, several carotenoid delivery systems are evaluated for their potential to decrease carotenoid degradation in functional food products. Keywords lycopene, lutein, beta-carotene, antioxidants

INTRODUCTION Carotenoids are the red, orange, and yellow pigments found in the photosynthetic tissues of plants, algae, and microorganisms, and they are responsible for the feather and esh colors of some birds and sh. Aside from coloration, these compounds are also important for plant and animal health. Carotenoids are tetraterpenes (Stahl and Sies, 1996) that are characterized by a conjugated system of double bonds with delocalized -electrons (Britton, 1995). Being extremely hydrophobic, these compounds are usually found in the core of membranes or other hydrophobic locations (Britton, 1995), where they are thought to function as antioxidants and play special roles in protection of tissues from damage caused by light and oxygen (Britton et al., 1995). Increasing awareness of potential health benets of certain carotenoids, has brought a surge of interest in creating functional food products containing carotenoid ingredients. However, the same properties that make carotenoids useful in healthy tissue function, create challenges in preventing the degradation of carotenoids in food products. In order to produce stable
Address correspondence to: Eric A. Decker, Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, Massachusetts 01003. E-mail: edecker@foodsci.umass.edu

carotenoid delivery systems for functional foods, ingredient developers must carefully consider the pathways that may lead to carotenoid degradation. By understanding these pathways, the predominant proxidants in the delivery system and the nal product can be identied and, in some cases, the delivery system can be modied to provide greater protection to the carotenoids. This review will discuss the health benets of several carotenoids that have received heightened interest in recent years, discuss the degradation mechanisms of carotenoids, and evaluate how several types of ingredient delivery systems might be engineered to protect these carotenoids. HEALTH BENEFITS OF CAROTENOIDS Several carotenoids commonly found in foods are thought to play a role in maintaining bodily functions and preventing disease. Beta-carotene, lycopene, lutein, and zeaxanthin are some of the most well known carotenoids considered to have health benets. Each of these compounds and their proposed benets are discussed below. Beta-Carotene

Beta-carotene (C40 H56 ) (Fig. 1) is one of the major carotenoids present in the diet (Johnson, 2002). It is found 515

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Figure 1

Structures of major carotenoids.

in a variety of orange, yellow, and green fruits and vegetables. Particularly good sources of beta-carotene include various greens (collard, turnip, spinach, lettuce), mangos, cantaloupe melons, peppers, pumpkin, carrots, and sweet potatoes (Holden et al., 1999). Beta-carotene, along with alpha-carotene and betacryptoxanthin, are sources of provitamin A. Once converted to vitamin A, health benets derived from these compounds include maintenance of normal eye health, epithelial function, embryonic development, and immune system function (NAS, 2001). Epidemiological studies have found that diets high in betacarotene or in fruits and vegetable intake are associated with decreased cancer risk (Peto et al., 1981; Vanpoppel and Goldbohm, 1995). These ndings led to the belief that beta-carotene may help reduce the risk of lung cancer (Johnson, 2002). However, several human intervention studies in which beta-carotene supplements were used actually found that beta-carotene increased the risk for lung cancer in high risk populations (smokers and asbestos-exposed workers) (Omenn et al.,1996; A-T, B-T, Cancer Prevention Study Group, 1994). These results have led to questions as to whether these effects were due to the high betacarotene dosage levels, the breakdown of beta-carotene into oxidation products, or if other components or a combination of components is responsible for the anticarcinogenic effects of fruit and vegetable consumption (Johnson, 2002; Palozza, 1998). In

addition to the debated anti-cancer benets of beta-carotene, this carotenoid has also been proposed to decrease risk factors for cardiovascular disease.

Lycopene Lycopene (C40 H56 ) is a hydrophobic, acyclic carotenoid containing eleven conjugated double bonds (Fig. 1) (Wilcox et al., 2003; Shi, 2000). Tomatoes, watermelon, guava, and grapefruit are the main sources of lycopene in the diet (Holden et al., 1999). In these raw plant products, approximately 95% of lycopene is found in the all-trans form (Shi, 2000; Nguyen and Schwartz, 1999), and is located in the photosynthetic pigmentprotein complex of the thylakoid membrane (Shi and Le Maguer, 2000). In some thermally processed foods and in human serum and tissues, higher quantities of cis isomers are found (Nguyen and Schwartz, 1999; Clinton, 1998). The popularity of lycopene as a bioactive food component has stemmed from a number of epidemiological studies that have concluded that diets rich in high lycopene foods are associated with a reduced risk of several diseases. In some cases, cell culture and dietary intervention studies have provided additional evidence that lycopene may play a positive role in health. Consumption of high lycopene foods has been proposed to reduce

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the risk of diseases including cardiovascular disease (Wilcox et al., 2003), and cancers of the prostate (Stahl and Sies, 1996; Hadley et al., 2002; Miller et al., 1996), breast (Shi, 2000), cervix, colon, esophagus, skin, pancreas, bladder, and stomach (Wilcox et al., 2003; Nguyen and Schwartz, 1999; Clinton, 1998). Of these conditions, the most evidence exists for lycopene and a reduced risk of prostate cancer.

Lutein and Zeaxanthin Lutein and zeaxanthin (C40 H56 O2 ) (Fig. 1) are oxygenated carotenoids, making them members of the xanthophyll group of carotenoids (Alves-Rodrigues and Shao, 2004). Lutein and zeaxanthin are stereo isomers, which complicates analytical techniques for determining the quantities of each stereo isomer present, and likewise, has created difculties in determining the inuence of the individual isomers on human health (Stacewicz-Sapuntzakis et al., 1993; Johnson, 2000). Particularly good sources of these carotenoids are leafy greens like spinach, collard greens, kale, corn, persimmons, and broccoli (Holden et al., 1999). Commercially produced lutein is derived from marigolds and is used in the poultry industry to impart yellow color to the yolks of eggs and the skin of broilers (Sowbhagya et al., 2004). Some evidence suggests that lutein and zeaxanthin are associated with a reduced incidence of age-related macular degeneration and cataracts as determined by epidemiological and intervention studies (Alves-Rodrigues and Shao, 2004; Sowbhagya et al., 2004; Richer, 2004). The mechanism by which these carotenoids are thought to decrease macular degeneration and cataract formation, is by increasing the macular pigmentation of ocular tissues, which helps to lter damaging blue light, thus preventing oxidation damage that eventually leads to tissue damage. (Alves-Rodrigues and Shao, 2004; Stringham and Hammond, 2005; Berendschot et al., 2000). However, other studies have not found a relationship between lutein and zeaxanthin and eye health (Mozaffarieh et al., 2003), indicating that more work is needed in this area of nutrition (Stringham and Hammond, 2005). While eye health is the predominant health benet associated with lutein and zeaxanthin, benecial effects of these carotenoids have been proposed for other health conditions as well. Lutein has been proposed to reduce risk factors for coronary heart disease and stroke, breast cancer, and improving skin health, although research in these areas is still limited (AlvesRodrigues and Shao, 2004; Johnson, 2000).

progression. There are still many questions over the exact chain of reactions responsible for these physiological actions. In addition, there are questions as to why certain carotenoids accumulate at high concentrations in some tissues of the body, but not others. For example, in humans, lycopene is found at low levels (0.22.4 nmol/g tissue) in ovarian, kidney, adipose, and liver tissues, while it is found to be high (2122 nmol/g tissue) in adrenal and testicular tissues (Kaplan et al., 1990). More research is needed to elucidate these questions of tissue specicity and mechanistic action. One of the mechanisms proposed for carotenoid protection of both plant and animal tissues is the quenching of singlet oxygen (Sies and Stahl, 1998). Singlet oxygen is capable of damaging both DNA and lipids within cells (Edge et al., 1997). In organic solvents, several carotenoids, including those already discussed, have been found to have exceptional ability to quench singlet oxygen (Edge et al., 1997; Conn et al., 1991; Baltschun et al., 1997). Carotenoids have also been found to act as free radical scavengers, reducing lipid oxidation reactions in certain model systems (Haila et al., 1997). Beta-carotene has been proposed to aid in the prevention of cardiovascular disease, possibly by protecting LDL cholesterol from oxidation or by reducing platelet aggregation (Gaziano and Hennekens, 1993), which would likely be due to the radical scavenging capacity of this compound. Another mechanism by which carotenoids may help to reduce the risk of certain cancers is by participating in gap junctional communication. This allows for cell-to-cell communication, which, when functioning properly, helps to inhibit the growth of altered cells (Stahl et al., 1997) that might otherwise generate tumors. Gap junctional communication has been found to be stimulated by beta-carotene (Stahl et al., 1997) and has also been proposed as a mechanism of action for lycopene (Sies and Stahl, 1998; Kun et al., 2006).

INFLUENCE OF ISOMERIC FORM ON BIOAVAILABILITY AND BIOACTIVITY Carotenoids are found in multiple isomeric forms due to the conguration of the long, conjugated system of double bonds characteristic of this group of pigments. The orientation of the double bonds is responsible for the shape and length of the molecule. There is some evidence that isomeric form may be an important factor in the bioactivity of certain carotenoids. For instance, cis-lycopene is found at high levels in human serum and tissues, while in most plant tissues and foods, the all-trans form predominates (Boileau et al., 1999). Unlike lycopene, the trans isomer of lutein is the predominant form found both in raw plant materials and some human tissues, and has also been found to be preferentially absorbed by cell cultures (Chitchumroonchokchai et al., 2004). The nding that cis-lycopene isomers concentrations are higher in animal tissues than in raw plant materials has created debate within the nutrition community as to whether cis

PROPOSED PHYSIOLOGICAL ACTIONS A number of physiological actions have been proposed for carotenoids. These actions have been theorized based on a combination of studies in model systems as well as a current understanding of how carotenoid chemistry might inhibit disease

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isomers may be preferentially absorbed by the body or translycopene is converted to cis once consumed (Clinton, 1998; Kun et al., 2006; Boileau et al., 1999, 2002; Unlu et al., 2007). Bile acid micelles mixed with lycopene isomers were found to have higher incorporation levels of cis isomers (Boileau et al., 1999). In the same study, lycopene was also fed to ferrets and it was found that signicantly higher levels of cis-lycopene isomers were present in lymph secretions than in stomach or intestinal contents, indicating that cis isomers were more bioavailable than all-trans isomers (Boileau et al., 1999). High plasma lycopene content results found in a trial using human subjects fed sauce made from tangerine tomatoes that have high levels (97%) of cis isomers add additional support to the hypothesis that cis lycopene isomers are more bioavailable than all-trans isomers (Unlu et al., 2007). Since all-trans lycopene is linear, it may be more likely to form into crystals that have lower solublity than its cis counterparts. In addition, the linear conguration of the all-trans lycopene might have difculty tting into bile micelles compared to curved cis forms thus making it difcult for the all-trans lycopene to be transported from the food to the intestinal microvilli from which they can be absorbed into the blood. Cis isomers of beta-carotene have also been found to have greater efciency of incorporation into micelles than all-trans beta-carotene in vitro (Levin and Mokady, 1995; Ferruzzi et al., 2006), but were not found to be preferentially absorbed by Caco-2 human intestinal cells (Ferruzzi et al., 2006). Difference in carotenoid solubility and shape may help explain their differences in bioavailability (Boileau et al., 1999). There is also evidence that thermal processing makes carotenoids more bioavailable. The reasoning behind this phenomenon is that the heating, increase in surface area, and agitation typically associated with thermal processing is likely to cause the breakdown of the cellular matrix of the plant material and may also induce trans to cis isomerization (Clinton, 1998; Dewanto et al., 2002; Schierle et al., 1997; Stahl and Sies, 1996; 1992; Agarwal et al., 2001; Xianquan et al., 2005).

nipulate the isomeric form of a lycopene ingredient to optimize solubility and other attributes inuencing bioavailability. Incorporating bioactive carotenoids into foods that do not naturally contain high amounts of these compounds may offer great health benets and provide new markets for food products. Addition of carotenoids to functional foods could be advantageous since they could be inporporated into an organic solution (i.e. an edible oil), which makes them more bioavailable than carotenoids in a plant cellular matrix. However, there are a number of stability issues that must be overcome before carotenoids can be successfully used as functional food ingredients since carotenoids placed in organic solutions, are less stable than carotenoids naturally occurring in tissues (Britton, 1995; Polyakov and Leshina, 2006). Therefore, it is essential to understand the mechanisms by which carotenoids may be degraded, in order to develop strategies for optimizing the stability of these bioactive ingredients.

CAROTENOID OXIDATION The conjugated polyene chain that is characteristic of carotenoids also makes these compounds susceptible to degradation from a number of agents. Depending on the carotenoid, the terminal end groups may also suffer degradation in certain environments. A number of carotenoid oxidizing agents have been identied. Carotenoid degradation pathways are highly inuenced by the agent involved in the initiation of degradation. Common oxidation mechanims are discussed below and summarized in Fig. 2. Once oxidation has been initiated by one of a number of oxidizing agents, carotenoids may further react with themselves or other chemical species within the environment to form a plethora of products. A summary of these products can be found in Fig. 3. In a functional food fortied with carotenoids, oxidative damage could result in loss of both product quality (color loss, rancidity, etc.) and bioactivity. These detrimental aspects of carotenoid oxidation highlight how it is important to have a critical understanding of the mechanisms of carotenoid oxidation so that technologies can be developed to optimize stability in functional foods.

CAROTENOIDS AS BIOACTIVE FOOD INGREDIENTS The evolving evidence that carotenoids can play a role in preventing disease has sparked interest in incorporating these compounds into functional foods. There is some evidence that carotenoids added to foods may be more bioavailable than their natural sources. For instance, some carotenoids, like lycopene can form crystalline aggregates in chromoplasts of plants cells (Shi and Le Maguer, 2000), which may lower their solubility and bioavalibility. Heat processing and the presence of lipids in the food matrix has been shown to improve the bioavalibility and trans-cis isomerization of beta-carotene and lycopene-rich foods, as would be expected because carotenoids are lipophilic and heat can induce isomerization (Stahl and Sies, 1996, 1992; Agarwal et al., 2001; Xianquan et al., 2005; Brown et al., 2004; Unlu et al., 2005). Theoretically, it might be possible to ma-

Autoxidation Reaction of carotenoids with atmospheric oxygen has been found to occur with relative ease, especially in systems consisting of puried carotenoids in organic solvents. Autoxidation of beta-carotene in benzene or tetrachloromethane in the dark at 30 C under one atmosphere of oxygen or by bubbling oxygen through the solvent was found to occur with an induction period of less than one hour, followed by rapid production of oxidation products. Under these conditions, beta-carotene was completely consumed within 30 hours. A combination of HPLC, FT-IR, and GC-MS were used to monitor the reaction and identify over twenty oxidation products (see Fig. 3). Further

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Figure 2

Mechanisms of carotenoid oxidation and initial products.

information about the reaction occurring in this system was gathered by adding AIBN (2,2 -azo-bis-isobutyronitrile (a free radical reaction initiator), BHT (butylated hydroxytoluene), or alpha-tocopherol to the system. Both BHT and alpha-tocopherol reduced the rate of oxidation product formation, while AIBN increased the rate of product formation. These results indicate that beta-carotene oxidation involves free radicals. The authors of this study propose that autoxidation may perhaps begin as beta-carotene in solution forms isomers via a biradical process (this may occur easily in solvent at 30 C), as seen in Fig. 4. The twisting of the molecule during the isomerization process may lead to an unpaired spin state, which can react easily with oxygen to form a carbon-peroxyl triplet biradicals. These may go on to form endo-peroxides or to react with a neutral beta-carotene molecule, forming an epoxide and a carotene alkoxyl radical. From the compounds detected in this study, it was concluded that the autoxidation process results rst in the production of epoxides, carbonyl compounds, and an uncharacterized oligomers, followed by further oxidation reactions of these compounds to produce secondary short chain carbonyl

compounds, carbon dioxide, and carboxylic acids (Mordi et al., 1993). Beta-carotene adsorbed to a C18 solid phase also exhibited autoxidation when oxygen-saturated water was owed continuously over the solid support. Using a combination of HPLC, UV-Vis spectroscopy, and electrospray LC-MS, a number of isomers and degradation products were detected. These included 13-cis, 9-cis and a di-cis isomers, beta-apo-13-carotenone, beta-apo-14 -carotenal, beta carotene 5,8-epoxide, and betacarotene 5,8-endo-peroxide (Henry et al., 2000). Retinoic acid, a compound derived from some provitamin A carotenoids (OByrne and Blaner, 2005), has been proposed to react directly with oxygen in the triplet state, at room temperature (Clark et al., 1997). This proposed pathway was based on the products formed when retinoic acid was exposed to 540 atm oxygen in 90% ethanol at room temperature. The products formed under these conditions were trans- and cis-5,8epoxy-5,8-dihydroretinoic acid, 5,6-epoxy-5,6-dihydroretinoic acid, 5,8-epidioxy-5,8-dihydroretinoic acid, 2-methyl-6-oxo2,4-heptadienal, beta-ionone, cyclocitral, dihydroactinidiolide,

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Figure 3 Overall oxidation products of beta-carotene oxidation.

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Figure 4

Possible pathways of the autoxidation of beta-carotene. Adapted from Mordi et al. 1993.

and 5-hydroxy-8-oxo-6,7-dihydroretinoic acid. 5,8-epidioxy5, 8-dihydroretinoic acid and 5-hydroxy-8-oxo-6,7-dihydroretinoic acid. These products were formed in the presence of peroxyl radical scavenger (2,6-di-tert-butyl-4-methylphenol) even though the formation of all other products was greatly inhibited, suggesting that these are products of direct oxygen addition while the other products produced without BMP present are due to concurrent autoxidation. The research group postulated that the mechanism of oxygenation may involve the collapse of electron donor-acceptor complexes forming carbon-peroxyl biradicals followed by intersystem crossing and ring closure (Clark et al., 1997).

Thermal Degradation Thermal treatment to carotenoids in the presence of oxygen results in the formation of volatile compounds and larger non-volatile components (Bonnie and Choo, 1999). Kanasawud and Crouzet (Kanasawud and Crouzet, 1990) proposed a sequence for beta-carotene degradation based on the products found during the heating of beta-carotene at 97 C for up to 3 hours in the presence of air as determined by

GC-MS and absorption spectrophotometry. They suggested that beta-carotene reacts with oxygen to form 5,6-epoxybeta-carotene, which can then convert to mutatochrome, 5,6,5 ,6 -diepoxy-beta-carotene, or luteochrome. Luteochrome may convert to aurochrome, which may then be cleaved to form dihydroactinidiolide. 2,5,6-epoxy-beta-carotene may cleave to form 5,6-epoxy-beta-ionone, which may be converted to betaionone, 2-hydroxy-2,6,6-trimethylcyclohexanone, 2,6,6-trimethylcyclohexanone, and 2-hydroxy-2,6,6-trimethylcyclohexane-1-carboxaldehyde. 2-Hydroxy-2,6,6-trimethylcyclohexane-1-carboxaldehyde can form beta-cyclocitral while 2-hydroxy-2,6,6-trimethylcyclohexanone can form 2,6,6trimethyl-2-cyclohexen-1-one (Kanasawud and Crouzet, 1990). Marty and Berset (1990), found that heating pure betacarotene at 180 C for two hours resulted in the formation of a number of cis isomers as well as oxidation products as determined by HPLC. This work also showed that the level of air circulation in the sample increased the degradation of betacarotene because of the greater likelihood of beta-carotene and oxygen interaction. The resulting compounds found in this work led the researchers to conclude that all of the double bonds of beta-carotene could be oxidized and that the breakage of

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bleaching of beta-carotene in some solvents like chloroform, can occur due to light exciting the beta-carotene molecules, which then instantly react with the solvent (chloroform in this case) to form either a carotenoid-solvent free radical adduct or a beta-carotene radical (due to hydrogen abstraction). The same work has also shown that the beta-carotene molecules in the excited state may return to ground state, where they may be attacked by radical by-products created during the above reaction and undergo a slower degradation process thought to possibly form beta-carotene radical cations (Mortensen and Skibsted, 1996).

Singlet Oxygen
Figure 5 Example of emulsion delivery systems that can be used in foods.

these bonds is likely to occur starting at the terminal end of the molecule and proceeding towards the center of the molecule. A study of beta-carotene in toluene (Handelman et al., 1991) conducted at 60 C with oxygen produced a complex mixture of products including, but not limited to epoxides as found by El-Tinal and Chichester. These products led the researchers to conclude that under these oxidation conditions, various radical species are formed and can react with oxygen to form peroxyl radicals, which can undergo propagation reactions with additional carotenoids (Handelman et al., 1991). Even under vacuum, higher temperature treatment (240 C) of crystalline beta-carotene resulted in the formation of toluene, m-xylene, pxylene, ionene, and 2,6-dimethylnaphthalene as determined by gas-liquid chromatography and infrared, nuclear magnetic resonance, and mass spectroscopies (Mader, 1964). At lower temperatures (60 C), with a stream of oxygen being passed through beta-carotene in toluene, El-Tinay and Chichester (1970), produced evidence that beta-carotene terminal double bonds could be broken, producing various epoxides as determined by chromatography followed by absorption spectra determination. This research showed that there was no lag phase in the decomposition of beta-carotene, ruling out autoxidation as a mechanism for degradation. They also showed that the reaction of oxygen with beta-carotene might be catalyzed by metals due to their ndings that cupric stearate increased the rate of reaction 4.3 fold (El-Tinay and Chichester, 1970). This research suggests that metals may have also been involved in the other autooxidation and thermal oxidation experiments as well since metal reactivity was not controlled in these studies. Photodegradation Light exposure degrades carotenoids, and several mechanisms of action have been proposed. Photooxidation produces species thought to be carotenoid radical cations (Konovalova et al., 2001; Mortensen and Skibsted, 1996). Laser ash photolysis studies have produced evidence to suggest that rapid

In a mechanism similar to carotenoid excitation described in photodegradation, light can also excite sensitizers like chlorophylls (Jung and Min, 1991), leading to the formation of singlet oxygen (1 O2 ). As seen in Eq. 1, singlet oxygen may then react with neutral carotenoids to produce excited state carotenoids (3 Car*). Once in an excited state, the carotenoid may return to ground state by releasing energy by vibrational and rotational interactions with the surrounding solvent (Eq. 2) (Stahl and Sies, 1996; Jung and Min, 1991; Krinsky, 1998; Choe and Min, 2006). The rate of singlet oxygen quenching in benzene at room temperature has been found to be 17, 13, 1212.6, and 6.6416 109 M1 s1 for lycopene, beta-carotene, zeaxanthin, and lutein respectively (Edge et al., 1997; Conn et al., (1991)
1

O2 + Car 3 O2 + 3 Car
3

(1) (2)

Car Car + Heat

Additionally, a computational study using density functional theory has concluded that while the physical quenching pathway described in Eqs. (1 and 2) is the most favored mechanism for carotenoid and singlet oxygen interaction, the excited state carotenoids may also follow a chemical degradation pathway that is still not well understood (Garavelli et al., 1998). This study suggests that the chemical pathway might involve a direct attack on the double bonds of the carotenoid by singlet oxygen, which forms biradicals that can eventually lead to carbonyl chain cleavage products (Garavelli et al., 1998). Yamauchi et al. proposed two products resulting from the chemical oxidation of carotenoids by singlet oxygen. In their work, beta-carotene 5,8endo-peroxide and beta-carotene 5,6-epoxide were produced during chlorophyll-sensitized photoxidation of beta-carotene in methyl linoleate (Yamauchi et al., 1998). Of these two products, it has been proposed that beta-carotene 5,8-endo-peroxide is the primary product, while beta-carotene 5,6-epoxide results from the creation of an oxygen-centered radical during reaction with singlet oxygen, which subsequently abstracts a hydrogen from the lipid medium and undergoes an intra-molecular hemolytic substitution reaction (SH i) reaction to form the nal product. Oxidation of beta-carotene with singlet oxygen was also found

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to produce beta-apo-14 -carotenal, beta-apo-10 -carotenal, betaapo-8 -carotenal, beta-ionone, and beta-carotene 5,8-endoperoxide as determined by HPLC, GC-MS, and LCMS/MS (Stratton et al., 1993). Chemical oxidation products were also found to form during the irradiation of lycopene in the presence of oxygen and methylene blue (sensitizer). These products were determined to be apo-6 -lycopenal, and a mixture of seven short-chain oxygenated compounds including 2-methyl2-hepten-6-one using MS, UV, IR, and 1 H- and 13 C-NMR spectra. The formation of these products led to the hypothesis that singlet oxygen, formed in the presence of the methylene blue sensitizer, reacted with lycopene to form a cyclic peroxide at C5 -C6 . Being a relatively unstable compound, this cyclic peroxide could easily undergo scission to form the detected products (Ukai et al., 1994).

al (Deng et al., 2000), 4 -apo-beta-caroten-4 -al (Deng et al., 2000), 8 -apo-beta-carotene-8 -nitrile (Deng et al., 2000), 6 apo-beta-carotene-6 -nitrile (Deng et al., 2000), 7 c-cyano-7 ethoxycarbonyl-7 -apo-beta-carotene (Jeevarajan et al., 1996), 7 ,7 -dimethyl-7 -apo-beta-carotene (Jeevarajan et al., 1996), and 4 -apo-beta-carotene-4 -nitrile (Deng et al., 2000) have all been degraded by iron, in studies using carotenoids and ferric chloride in dichloromethane (Jeevarajan et al., 1996; Wei et al., 1997; Deng et al., 2000; Gao et al., 1997, 2003, 2003; Polyakov.et al., 2001), or in sol-gels containing iron in the aqueous fraction (He and Kispert, 2001). The proposed interaction of neutral carotenoid with ferric iron can be seen in Eq. 4. Car+ + Fe3+ Car + +Fe2+ (4)

Acid Exposure to acids is thought to produce ion-pairs, which can then dissociate to form a carotenoid carbocation. This process can be seen in Eq. 3 (Konovalov and Kispert, 1999). Car + AH (CarH+... A ) CarH+ + A

(3)

Optical spectra of carotenoid carbocations have been reported for beta-carotene, 8 -apo-caroten-8 -al, and canthaxanthin when exposed to triuoroacetic acid in benzene, CH2 Cl2 , and acetonitrile solution (Konovalov and Kispert, 1999). He and Kispert (2001) showed that canthaxanthin and 8 -apo-betacaroten-8 -al incorporated into sol-gels could be degraded by sulfuric acid (pH 3-3.5).

Iron and Iodine Some evidence indicates that iron may be capable of directly interacting with carotenoids to produce degradation products. In most work completed to date, ferric chloride is the commonly used iron oxidizing agent for studying the degradation of carotenoids (Konovalova et al., 2001, 2000; He and Kispert, 2001; Jeevarajan et al., 1996; Wei et al., 1997; Deng et al., 2000; Gao et al., 1997, 2003, 2003, 2006; Polyakov et al., 2001; Ding et al., 1988). Beta-carotene (He and Kispert, 2001; Jeevarajan et al., 1996; Konovalova et al., 2000; Gao et al., 1997, 2003, 2003; Polyakov et al., 2001), canthaxanthin (He and Kispert, 2001; Jeevarajan et al.,1996; Konovalova et al., 2000; Wei et al., 1997; Gao et al., 1997, 2003; Polyakov et al., 2001), 8 -apobeta-caroten-8 -al (He and Kispert, 2001; Konovalova et al., 2000; Wei et al., 1997; Deng et al., 2000; Polyakov et al., 2001), ethyl 8 -apo-beta-caroten-8 -oate (Konovalova et al.,2000; Deng et al., 2000; Gao et al., 2003; Polyakov et al., 2001), 7 apo-7 ,7 -dicyano-beta-carotene (He and Kispert, 2001), ethyl6 -apo-beta-caroten-6 -oate (Deng et al., 2000), ethyl 4 -apobeta-caroten-4 -oate (Deng et al., 2000), 6-apo-beta-caroten-6 -

The reaction results in the formation of a carotenoid cation radical and ferrous iron as determined by comparison of the optical (Jeevarajan et al., 1996; Konovalova et al., 2000; Deng et al., 2000; Gao et al., 1997, 2003), and stop-ow absorption spectra (Deng et al., 2000) to the spectra produced from oxidation of carotenoids in cyclic voltammetry, bulk electrolysis (Jeevarajan et al., 1996; Wei et al., 1997; Deng et al., 2000; Gao et al., 1997). EPR spectroscopy (Konovalova et al., 2000), or square voltammetry (Deng et al., 2000; Gao et al., 2006) studies. This reaction can occur in the presence or absence of near-UV to visible light. However, irradiation with near-UV to visible light increases reaction rates (Konovalova et al., 2000; Gao et al., 1997). When light is present, solutions of carotenoids (specically beta-carotene and canthaxanthin) and ferric chloride in dichloromethane have regenerated ferric iron from the ferrous iron produced in Eq. 4. The reproduced ferric iron can go on to react the with the remaining neutral carotenoid or may react with carotenoid cation radicals produced in Eq. 4 to form dications as seen in Eq. 5 (Jeevarajan et al., 1996; Gao et al., 1997) Car + + Fe3+ Car2+ + Fe2+ (5)

Gao and Kispert (2003) found evidence of dication formation by examining UV/vis spectra and Osteryoung square-wave voltammetry results after the addition of 4 equivalents of FeCl3 to ethyl-all-trans-8 apo-beta-caroten-8 -oate or beta-carotene in dichloromethane. The production of carotenoid dications may also be concentration dependent with respect to iron. Increasing levels of ferric chloride have been found to result rst in formation of cation radicals (0.5 equivalents ferric chloride). With additional ferric chloride (2 or more equivalents), dication carotenoids were formed from beta-carotene, 7 -cyano-7 -ethoxycarbonyl7 -apo-beta-carotene, canthaxanthin, or 7 ,7 -dimethyl-7 -apobeta-carotene. The transformation to a cation or dication alters the optical absorption spectra of the carotenoid. For all carotenoids tested in this study, the max for the three species increased in the order of neutral carotenoid < dication < cation radical (Jeevarajan et al., 1996).

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Iodine is a milder oxidizing agent than ferric chloride (Jeevarajan et al., 1996), yet, it has been found to degrade 8 -apobeta-caroten-8 -al, 7 -apo-7 ,7 -di-cyano-beta-carotene, ethyl 8 apo-beta-caroten-8 -oate, canthaxanthin (Konovalova et al., 2000) and beta-carotene to cation radicals as determined by UV-vis and EPR spectroscopy (Konovalova et al., 2000). Reaction of pure beta-carotene with 123 I in benzene and light petroleum resulted in beta-carotene-tri-iodide as determined by elemental analysis and M ossbauer spectroscopy. This product is formed by a one-electron transfer from beta-carotene to triiodide (Matsuyama et al., 1982).

and radical. For instance, there is evidence from laser ash and steady-state photolysis studies that beta-carotene is not likely to interact with peroxyl radical in this manner, but instead are more likely to undergo adduct formation or hydrogen abstraction reactions (discussed in sections 2.3.7.2 and 2.3.7.3) (Mortensen and Skibsted, 1988). ROO +Car ROO + Car+ (6)

Free Radicals In some systems, such as foods, free radicals may already be present in the medium due to reactions such as lipid oxidation (Choe and Min, 2006). Carotenoids have been found to confer both antioxidant and prooxidant effects in systems containing pre-formed radicals depending on the type and level of carotenoid used, oxygen concentration present, and the polarity of the solvent (Haila et al., 1997). When radicals are present in a system, several mechanisms of carotenoid interaction are possible. These reactions include electron transfer, hydrogen abstraction, and addition of radical species to form carotenoidradical adducts (see Fig. 2). (Britton, 1995; Young and Lowe, 2001; Mortensen, 2002). The initial products of electron transfer, hydrogen abstraction, and adduct formation reactions, can in turn react with additional species via a number of mechanisms, including other radical interactions. A variety of products, summarized in Fig. 3, result from these secondary reactions. Each of the three radical interaction mechanisms, and the subsequent reactions of the resulting products are described below.

If carotenoid radical cations are formed, a number of reactions may occur. One path they may follow is to interact with oxygen to form a carotenoid peroxyl radical cation (Eq. 7), which might then be reduced by another carotenoid or ferrous iron, if present, to create a peroxide form of the carotenoid (Eqs. 8 and 9) (Gao et al., 2003). In one study, this series of reactions was determined to produce high amounts (90% yield) of the 5,8-endo-peroxide of both all-trans-beta-carotenene and ethyl all-trans-8 -apo-beta-caroten-8 -oate as determined by APCI LC-MS and 1 H NMR (Gao et al., 2003). If iron is present, reactions previously covered in discussion of Eq. 5, may occur. One study has also found that bulk electrolysis of canthaxanthin in CH2 Cl2 with ferric chloride at low temperature (10 C) followed by irradiation with UV-vis light for 1.5 minutes, can lead to the formation of carotenoid dimers. This is thought to occur by the formation of a radical cation that then reacts with neutral carotenoids to form dimers under these conditions (Gao et al., 1977). Pulse radiolysis studies have also found radical cations to decay in a bimolecular process, although the products were not determined (Mortensen et al., 1997). Car + +O2 CarO2 + CarO2 + +Fe2+ CarO2 + Fe3+ CarO2 + +Car CarO2 + Car+ (7) (8) (9)

Electron Transfer Some studies suggest that neutral carotenoids are capable of participating in electron transfer reactions with radicals as well as with metals like iron as discussed previously. In these reactions, carotenoid radical cations are formed (Eq. 6) (Britton, 1995; Gao et al., 1997, 2003; Young and Lowe, 2001; Burke et al., 2001; Mortensen et al., 1997). Beta-carotene, canthaxanthin, zeaxanthin, astaxanthin, and lycopene have been shown to form radical cations by electron transfer reactions with tryptophan radical cations in a pulse radiolysis study using carotenoids in micelles (Burke et al., 2001). Pulse radiolysis has also shown that radicals of nitrogen dioxide react with carotenoids in this manner in lycopene, lutein, zeaxanthin, astaxanthin, or canthaxanthin in tert-butanol/water mixtures. The same reaction mixtures and pulse radiolysis techniques also found thiyl-sulphonyl radicals to produce carotenoid radical cations, but an uncharacterized intermediate (possibly and ion-pair) was detected as well (Mortensen et al., 1997). The likelihood of electron transfer reactions taking place may depend on the type of carotenoid

A nal reaction series that carotenoid radical cations have been found to undergo in solvent systems is deprotonation. This reaction has been studied using canthaxanthin and betacarotene in dichloromethane using electrochemistry and optical, MALDI-TOF, and EPR spectroscopy (Gao et al., 2003). The deprotonation reaction (Eq. 10) was found to be enhanced by the presence of water in the solvent system (Gao et al., 2003). Density functional theory applied to this reaction has led to the conclusion that for beta-carotene radical cations, deprotonation at the 5 or 5 methyl group on the cyclohexene ring would result in the most stable product (Gao et al., 2006). Car+ #Car +H+ #Car +O2 #Car OO (# indicates one less proton) (10) (11)

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It has been suggested that deprotonated carotenoid radicals may be capable of reacting with oxygen to form additional radicals (Eq. 11) (Konovalova et al., 2000). In addition, the deprotonated radical has been found to react with other deprotonated radicals to form didehydrodimers (Eq. 12) (Gao et al., 2003). #Car +#Car (#Car)2 (12)

As already discussed, carotenoid dications may be formed by reaction of carotenoid radical cations with iron (Eq. 5). Electrochemical oxidation of beta-carotene has also been shown to produce dications in tetrahydrofuran, dichloromethane and dichloroethane solutions as determined by EPR (Grant et al., 1988). Whether radical cations or dications are the predominant species formed, may depend on the structure of the carotenoid. Khaled et al. (1991) found that beta-carotene and beta-apo-8 -carotenal form predominantly dications in experiments using simultaneous electrochemical-EPR of carotenoids in dichloromethane, while canthaxanthin produced predominantly radical cations. If dications are produced, two additional decay reactions have been proposed. The dication may be able to react with a neutral carotenoid in the system, creating two radical cations (Eq. 13) (Khaled et al., 1990), or it may be deprotonated (Eq. 14), in a pathway like that shown in Reaction 10, which is especially enhanced in the presence of water (Jeevarajan et al., 1996; Deng et al., 2000; Gao et al., 1997; Khaled et al., 1990). Reactions (13 and 14) were studied by Khaled et al. using EPR and cyclic voltametry (Khaled et al., 1990). This work found evidence that dications of beta-carotene, canthaxanthin, and beta-apo-8 -carotenal in dichloromethane or dichloroethane can undergo both Reactions 13 and 14. A mixture of carotenoid radical cations and dications was produced using varied amounts of ferric chloride (1-2 equivalents) to oxidize canthaxanthin in dichloromethane, suggesting that Reaction 13 was occurring in this system (Jeevarajan et al., 1996). Car2+ + Car 2Car+ Car2+ #Car+ + H+ (13) (14)

can be seen in Eqs. 15 and 16 (Choe and Min, 2006; Liebler and McClure, 1996; Woodall et al., 1997). Liebler and McClure proposed this as a potential mechanism by which beta-carotene oxidation products were formed during oxidation with AMVN (2,4-dimethylvaleonitrile) radicals in benzene at 60 C based on atmospheric pressure chemical ionization mass spectrometry results (Liebler and McClure, 1996). Woodall et al. also proposed Reaction 15 as a mechanism by which beta,beta-carotene might react with peroxyl radicals, postulating that the allylic C-4 position of the molecule might be the site of attack. This hypothesis was based on electron density calculations and UV-vis and mass spectrophotometry results produced during the reaction of beta,beta-carotene with AIBN radicals in the presence of methanol (Woodall et al., 1997). Mortensen and Skibsted also proposed this mechanism as well as adduct formation as possible means by which alkyl, alkoxyl and alkylperoxyl radicals might react with beta-carotene (Mortensen and Skibsted, 1997). ROO +Car ROOH + Car OH +Car H2 O + Car (15) (16)

It has been proposed that the newly formed carotenoid radical might then encounter an additional alkoxyl or peroxyl radicals in the medium and react to form a non-radical product (Eqs. 17 and 18) (Choe and Min, 2006; Liebler and McClure, 1996). Car +RO Car-OR Car +ROO Car-OOR Adduct Formation Finally, radicals can also react with carotenoids to form radical adducts. This may occur with alkyl, alkoxyl, and peroxyl radicals as seen in Eqs. 1921 (Haila et al., 1997; Mortensen, 2002; Liebler and McClure, 1996). Laser ash and steadystate photolysis studies of beta-carotene indicate that at least for this carotenoid, reaction with peroxyl radicals occurs much more slowly than with alkyl or alkoxyl radicals (Mortensen and Skibsted, 1988). Iannone et al. showed that beta-carotene and lutein can quench peroxyl radicals at low oxygen concentrations using EPR and spin trapping techniques (Iannone et al., 1998). Laser ash photolysis studies of beta-carotene and canthaxanthin in aerated solutions of benzene with di-tert-butyl peroxide and toluene have provided evidence that the benzylperoxyl radical produced under these conditions reacts with the carotenoids to produce adducts that decay in a rst-order reaction (Mortensen, 2002). Acetylperoxyl radicals produced during laser ash photolysis have also been shown to produce adducts with beta-carotene in aerated benzene at 20 C. The second-order rate constant of this reaction is 9.2 108 M1 s1 (Mortensen, 2001). Pulse radiolysis has also shown that glutathione thiyl radicals and 2-mercaptoethanol thiyl radicals react with carotenoids (17) (18)

The deprotonation reaction (Eq. 14) has been studied using electrochemistry (Gao et al., 2003), optical spectroscopy (Gao et al., 1997, 2003). MALDI-TOF spectroscopy (Gao et al., 2003), and EPR spectroscopy (Gao et al., 2003), using canthaxanthin (Gao et al., 1997, 2003), and beta-carotene (Gao et al., 1997, 2003).

Hydrogen Abstraction Reaction of neutral carotenoid with a radical can result in the radical abstracting a hydrogen from the carotenoid. This results in the formation of a resonance stabilized carotenoid radical as

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in a rst-order reaction to produce adducts followed by bimolecular decay of the adducts in solutions of 10 M lycopene, lutein, zeaxanthin, astaxanthin or canthaxanthin in tert-butanol/water mixtures (Mortensen et al., 1997) Car + R (Car-R) Car + RO (Car-RO) Car + ROO (Car-ROO) (19) (20) (21)

is reduced. At higher oxygen pressures, however, the reaction is driven to the right and autoxidation rates increase due to hydrogen abstraction reactions like that shown in Eq. 25. ROO-Car-OO +RH ROO-Car-OOH + R (25)

Reaction with additional radicals is thought to result in addition products. Reaction 22 was proposed to be the mechanism by which beta-carotene addition products, detected by atmospheric pressure chemical ionization mass spectrometry, formed in reaction with AMVN-derived alkyl and alkoxyl radicals (Liebler and McClure, 1996). The addition product in Eq. 23 was suggested to be the main product of beta-carotene and lutein oxidation at low oxygen concentrations (Iannone et al., 1998) (Car-RO) +RO RO-Car-RO (Car-ROO) +ROO ROO-Car-ROO (22) (23)

An additional pathway that radical adducts might follow involves reaction with oxygen. In what is thought to be a reversible reaction, oxygen may add to the radical adduct, producing a resonance stabilized, chain carrying peroxyl radical (Eq. 24) (Burton and Ingold, 1984). Liebler and McClure (1996) proposed this as as well as hydrogen abstraction as potential mechanisms by which beta-carotene oxidation products were formed during oxidation with AMVN radicals in benzene at 60 C based on atmospheric pressure chemical ionization mass spectrometry results. El-Agamey and McGarvey (2005) were the rst to observe reversible oxygen addition to neutral 7,7 -dihydrobeta-carotene radicals and neutral beta-carotene radicals utilizing phenylthiyl radicals produced by laser ash photolysis. The rate constants determined for the addition of oxygen to these neutral radicals were determined to be 0.64 104 M1 s1 for beta-carotene radicals and 4.3 104 M1 s1 for 7,7 -dihydrobeta-carotene radicals (Burton and Ingold, 1984). (Car-ROO) +O2 ROO-Car-OO (24)

Peroxyl radical adducts, however, are also likely to follow other pathways. One path these radical adducts might follow is decay to carotenoid epoxides and new alkoxyl radicals (Eq. 26), which may further the extent of oxidative degradation as the newly formed radicals attack other oxidizable substrates in the system (Liebler and McClure, 1996). Mortensen (2001) found acetylperoxyl-beta-carotene radical adducts to decay with a rst order rate constant of 1.35 103 s1 , and hypothesized that the decay of the adduct may follow this path (Mortensen, 2001) Yamauchi et al. (1993) found additional evidence for this type of pathway in the reaction products formed by AMVN-initiated peroxidation of beta-carotene in methyl linoleate or benzene. The products formed in the benzene study (as determined by HPLC followed by UV, infrared, 1 H and 13 C NMR and mass spectrometry) were 12-formyl-11-nor-beta, beta-carotene, 15 -formyl-15-nor-beta, beta-carotene, 19-oxomethyl-10-norbeta, beta-carotene, 5,6-epoxy-5,6-dihydro-beta, beta-carotene, beta-carotene, 13,15 -epoxyvinyleno-13,15 -dihydro-beta, stereoisomers of 15 ,13-epoxyvinyleno-13,15 -dihydro-beta, beta-carotene, and 11,15 -cyclo-12,15-epoxy-11,12,15,15 tetrahydro-beta,beta-carotene (Yamauchi et al., 1993). In the methyl linoleate study, products formed (as determined by HPLC) included beta-carotene 5,6-epoxide, 13,15 -epoxyvinyleno-13,15 -dihydro-14,15-dinor beta,betacarotene, 15 ,13-epoxyvinyleno-13,15 -dihydro14,15-dinorbeta,beta-carotene, 11,15 -dihydrooxepin-beta-beta-carotene, 12-formyl-11-nor-beta, beta-carotene, and 15 -formyl-15-norbeta, beta-carotene (Yamauchi et al., 1998) (Car-ROO) Carotenoid Epoxide + RO (26)

ISOMERIZATION Electron transfer reactions are also thought to play a role in isomerization of carotenoids. Some studies suggest that isomerization is mediated by carotenoid radical cations and possibly dications (Eqs. 27 and 28) formed from electron transfer from neutral carotenoids to radicals or compounds like iron that can easily transfer charge (Wei et al., 1997; Gao et al., 2003, 1996). This is thought to occur because of lower energy barriers for congural transformation in the cation species compared to those of neutral carotenoids (Wei et al., 1997; Gao et al., 1996), Wei et al. (1997) found that oxidation of canthaxanthin and 8 -apo-betacaroten-8 -al resulted rst in the formation of radical cations (as determined by optical spectroscopy), followed by formation of cis-isomers (as determined by HPLC). Canthaxanthin and betacarotene in dichloromethane were also found to undergo this

As rst described by Burton and Ingold (1984), the reaction described in Eq. 24 is likely governed by oxygen pressure. In experiments with beta-carotene, rates of carotenoid autoxidation were found to decrease when oxygen levels were lowered. Similarly, as oxygen levels were lowered , beta-carotene had a greater antioxidant effect in the AIBN-initiated oxidation of Tetralin and methyl linoleate in chlorobenzene at 30 C. These results led Burton and Ingold to conclude that as oxygen is lowered (often to levels similar to those in animal tissues), Reaction 24 shifts to the left and production of peroxyl radicals

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process as determined by bulk electrolysis with simultaneous absorption spectroscopy (Gao et al., 1996) trans -Car+ cis -Car + trans -Car2+ cis -Car2+ (27) (28)

The cis-radical cations and dications are then thought to undergo reactions with neutral trans-carotenoids still present in the system to form either neutral cis-carotenoid or cis-radical cations as well as new trans-radical cations that can be recycled through the isomerization reaction sequence (Eqs. 29 and 30) (Wei et al., 1997; Gao et al., 1999) cis -Car + +trans -Car cis -Car + trans -Car+ cis -Car2+ + trans -Car cis -Car + +trans -Car+ (29) (30)

Isomerization has also been found to occur during the oxidation of ethyl 8 -apo-caroten-8 -oate and beta-carotene by ferric chloride. This study proposed that if iron is present in the system, ferrous iron can react with the cis-radical cations or dications and be oxidized to ferric iron and either the neutral cis-carotenoid or the cis-radical cation, just as seen in the reaction with neutral trans-carotenoid in Eqs. 29 and 30 (Gao et al., 2003) . Isomerization is also thought to occur if carotenoids are exposed to acids. Similar to the carotenoid radical cation formed in electron transfer based isomerization, a carotenoid carbocation (CarH+ ) is believed to be an intermediate in trans-cis isomerization. This carbocation has been predicted by Austin Model 1 (AM1) calculations of rotation barriers to have a lower barrier to rotation than neutral carotenoids, facilitating the isomerization process (Konovalov and Kispert, 1999).

DELIVERY SYSTEMS FOR CAROTENOIDS Understanding the mechanisms of carotenoid degradation is essential for developing technologies for the incorporation of these compounds into functional foods. Carotenoids could be added to foods as pure compounds, oleoresins of foods (e.g. paprika), or as dried food products (e.g. tomatoes). While these options may be viable in some foods, they may be limited in others by problems with solubility, avor, and stability. An alternate possibility for incorporating carotenoids into foods would be to incorporate them into emulsion or nanostructure delivery systems that if necessary could be further encapsulated by drying operations. These dispersed forms of carotenoids would have the advantage of being more easily dispersed into food products. In addition, delivery systems could be designed to help decrease the degradation of the carotenoids. By identifying the predominant carotenoid degradation pathway likely to occur in a particular food product, delivery systems could be engineered for optimal stability.

For carotenoid delivery systems, several concerns arise when considering common prooxidants that might be present once a carotenoid ingredient delivery system is incorporated into a food product. One major issue is likely to be the presence of iron. Iron is ubiquitous in food products, creating a need to limit contact between carotenoids. Strategies to control contact include the ability to create a physical or electrostatic barrier between the carotenoid and iron or by adding a chelator to bind the iron, partitioning it away from the carotenoids or making the iron less reactive. Another set of prooxidants of concern are radical species. Again, radicals are likely to be present in food products due to reactions such as those associated with lipid oxidation. To limit the presence of radicals, delivery systems are needed that can allow for the incorporation of multiple types of free radical scavengers. It might also be possible to formulate delivery systems in such a way that radical production is limited, by selecting more saturated lipid sources that are less likely to oxidize. Finally, it may be important to prevent contact with light to prevent photodegradation of carotenoids. In addition to chemical stability concerns, several other factors need to be considered in creating a delivery system for carotenoids. The delivery system needs to be one in which the carotenoid ingredient can be easily dispersed into multiple types of food products. If possible, it is desirable that any protection provided to carotenoids in the delivery system continues to offer protection once the ingredient is incorporated into the food. It is also important to consider whether the delivery system will alter the appearance, avor, or texture of the food product. Finally, the bioactivity and bioavailability of the carotenoid, and any desire to control the release of the carotenoid during digestion should be determined (McClements et al., 2007). Several systems including conventional emulsions, multilayer emulsions, and solid lipid particles have some of the desirable characteristics for carotenoid delivery systems. Each of these systems has different advantages and disadvantages depending on the oxidation pathways of concern as well as any formulation and processing requirements of the nal food product that might lead to physical instability of the system.

Conventional Emulsions Since carotenoids are primarily lipid-soluble, one way to incorporate them into foods is in the oil phase of oil-in-water emulsions. These emulsions are typically produced by homogenizing oil and aqueous phases along with emulsiers at high pressure. This process results in oil droplets coated in the surfactant that form an interfacial layer between the oil and aqueous phases (Fig. 5) (McClements, 2005). Emulsions have been found to be an effective way of incorporating bioactive lipids into food products. For example, emulsions were used to create a delivery system for omega-3 fatty acids in ice cream and yogurt. These emulsions were found to be physically and oxidatively stable, with little consumer detection of altered sensory attributes (Djordjevic et al., 2004; Chee et al., 2007, 2005).

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These studies suggest that emulsions also be useful for delivering other lipid-soluble bioactive components. Emulsions have several advantages as potential carotenoid delivery systems. All three regions of the emulsion may be engineered to add stability. By carefully selecting the emulsier used in the preparation, the interfacial region formed can create physical or electrostatic barriers to iron and other prooxidants that are common to the aqueous components of foods. The oil in which the carotenoid is dispersed may be selected to improve oxidative stability. For instance, more saturated oils may have greater stablity to lipid oxidation, thus reducing the potential for the production of radical species that could in turn react with carotenoids. Oils may also contain natural or added antioxidants such as tert-butylhydroquinone, butylated hydroxytoluene, tocopherols, and avonoids. Hydrophilic components, including free radical scavenging antioxidants (ascorbic acid, phenolics, peptides, or proteins) and chelators (EDTA, citric acid, polyphosphates, and proteins) may be added to the aqueous phase to reduce interactions with iron or reduce the presence of radical species that could destroy carotenoids. The pH of the environment may also be controlled by the formulation of the aqueous phase, which may help to prevent acid-initiated degradation as well as controlling the solubility of aqueous components like iron that is known to degrade carotenoids. Emulsions are easy to incorporate into aqueous based products at a relatively low cost and they could retain their antioxidant properties once diluted into the food if their chemical and physical properties are retained. An additional advantage of using emulsions as a delivery system for lipid-soluble bioactives, is that the emulsion system can contain high amounts of bioactives without having high lipid content, which may be more attractive to health-consious consumers attempting to limit fat in their diet. While emulsions have the potential to offer many protective characteristics as carotenoid delivery systems, they also have limitations. A serious problem found in using emulsions is that they have a tendency to become unstable when subjected to environmental stresses commonly encountered in food processing (heating, freezing, altering pH) (McClements, 2005). An additional problem is that the high surface area of emulsied lipids could create a large contact area that could increase interactions between the carotenoids in the lipid droplet and water-soluble prooxidants.

Multilayer Emulsions Multilayer emulsions are produced by forming layers around an oil droplet (Fig. 5). Typically, a conventional emulsion is made followed by addition of a polyelectrolyte of opposite charge (e.g. polysaccharide or protein). The opposite charges attract to one another, causing the polyelectrolyte to adsorb to the droplet surface. This process can be repeated, again using

the principle of electrostatic attraction to form additional layers (Guzey et al., 2004). Multilayer emulsions retain all of the advantages previously described for conventional emulsions. In addition, the thicker interfacial membrane present in these emulsions may provide added protection to carotenoids from prooxidants in the aqueous phase of the product. These prooxidants may include iron, acids, and radical species. Depending on the composition and thickness of the interfacial membrane, there may also be a reduction in the amount of light reaching the carotenoids in the oil droplets. Another advantage of multilayer emulsions is that the layers can be engineered to release from the droplet under certain environmental conditions. Therefore, these delivery systems could be created in such a way that they could allow for triggered release of carotenoids during digestion (McClements et al., 2007). This might possibly reduce the degradation of carotenoids during the digestive processes prior to absorption, and may improve bioavailability. Multilayer emulsions have been successfully used to increase the oxidative stability of omega-3 fatty acids compared to conventional emulsions. In a comparison of spray-dried conventional lecithin-stabilized menhaden oil emulsions to multilayer lecithin and chitosan-stabilized mehaden oil emulsions, propanal (a lipid oxidation product) was detected in the lecithin only emulsion within four days, while the emulsion with the additional chitosan layer did not exhibit propanal production for seven days. In this work, the emulsions were spray dried and re-dispersed into a liquid. Upon reconstituting with liquid, the multilayer system remained intact and was able to inhibit oxidation, suggesting that multi-layer emulsions would not only be effective as liquid ingredients but also as spray-dried powders (Shaw et al., 2007). Colloidosomes, a special type of multilayer emulsion, might also offer advantages for protecting carotenoids solubilized in a lipid carrier. Colloidosomes are produced by using electrostatic attraction to coat an oil droplet with smaller droplets of a second emulsion made with a surfactant of opposite charge to the one used to make the larger primary emulsion (Gu et al., 2007). As with multilayer emulsions formed with biopolymers, colloidosomes have the advantages of forming a thick physical barrier to inhibit contact with aqueous prooxidants, and may be formulated in such a way as to allow for controlled release of carotenoids. In addition, the smaller droplets used to coat the carotenoid-containing, larger droplet could be loaded with concentrated antioxidants to protect carotenoids in the inner droplet. This might prove especially useful since the antioxidants would be concentrated at the site of the prooxidant attack rather than being diluted throughout the system. The main concern in using multilayer emulsions as delivery systems is that careful control over processing parameters is needed to ensure the physical stability of the droplets (Guzey et al., 2004). These systems are also likely to have higher production costs than conventional emulsions due to the additional processes and ingredients needed to produce them.

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Solid-Lipid Particles Solid-lipid particles can be produced using the similar methods to those used for creating conventional emulsions. However, the lipid components are carefully chosen so that all or part of the lipid in the emulsion droplets solidies after processing and cooling. A key component to solid-lipid particle production is to homogenize the lipid and aqueous phases at a temperature that is higher than the melting point of the lipids (Helgason et al., 2008; Schubert and Muller-Goymann, 2005). If the temperature is too low, solid lipid components will not be homogenized and may damage the processing equipment (McClements et al., 2007). After processing, cooling of the formed particles is also important to form a solid lipid emulsion droplet with the correct lipid crystal conguration (Fig. 5). By choosing multiple lipid types and controlling cooling, it is possible to form multiple types of solid-lipid particles. Coreshell structures can be made to give an outer shell of one lipid and an inner core of another lipid. Depending on the melting points of the chosen lipids, these methods can produce coreshell particles with solid outer shells and liquid inner cores, liquid outer shells and solid cores, or solid shells with solid cores. It is also possible to produce solid-lipid particles with one lipid randomly dispersed in another lipid. Again, in these particles, one component can be solid while the other lipid is liquid (McClements et al., 2007). There are several potential advantages to using solid-lipid particles for delivering carotenoids into foods. First, incorporation of carotenoids into the core of core-shell solid-lipid particles, may offer a physical barrier for protecting carotenoids from aqueous prooxidants. Second, the nature of the lipids used to create these particles may help to decrease the rate of oxidation reactions. Since more solid-like lipids are needed to form solid particles, it is likely that the lipids chosen for solid-lipid particle applications will be more saturated than fats commonly used in conventional emulsions. Saturated fats undergo oxidation more slowly than unsaturated fats (Nawar, 1996), potentially lowering the likelihood that carotenoids would undergo radical attack from radicals produced in lipid oxidation reactions. Additionally, solid-lipid particles may be useful in creating physically stable systems for the delivery of carotenoids like lycopene, that often form crystal structures. In conventional emulsions, the presence of crystals can create physical instability by causing partial coalescence of the lipid droplets (McClements, 2005). However, in solid-lipid particles, it may be possible to overcome this problem by dispersing the crystalline carotenoid into a larger solid lipid phase. Finally, solid-lipid particles offer the potential to allow for the controlled release of carotenoids in the inner core of the droplet by selecting a lipid for a solid outer shell that has a melting point around the temperature of the environment where carotenoid release is desirable (human body temperature, during nal food preparation, etc.). Although there are many potential advantages to solid-lipid particles as incorporation systems for carotenoids, drawbacks remain. Since heating is required to form solid-lipid particles,

there is potential for thermal degradation of carotenoids during this process. In addition, the more saturated lipids needed to produce these particles may be unattractive to health conscious consumers trying to reduce their intake of saturated fats. Bioavailability of incorporated carotenoids may also be of concern. Crystalline structures of carotenoids may be less soluble and less bioavailable than non-crystalline carotenoids, which may make solid-lipid particles with dispersed carotenoid crystals less benecial. Finally, if lipid crystallization is not done properly, the carotenoid could end up concentrated on the outside of the lipid core where it could be more susceptible. This problem was seen in solid emulsion droplets containing omega3 fatty acids (Okuda et al., 2005).

CONCLUSIONS As further evidence on the health benets of carotenoids is discovered, there is likely to be an increased demand for the addition of carotenoids into functional food products. However, due to the instability of carotenoids and multiple mechanisms of carotenoid degradation in foods, it is essential that the mechanisms of carotenoid degradation in each product are identied and that ingredient formulations are engineered to ensure carotenoid stability over the shelf-life of the food. Conventional emulsions, multi-layer emulsions, and solid-lipid particles are several carotenoid delivery systems that should be considered for use in functional food formulations since each of these systems has the potential to offer multiple forms of carotenoid protection, while still allowing for easy incorporation into foods.

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